Entomology Research Institute, Loyola
College, Chennai,

Aerial parts and
flowers of Sphaeranthus indicus
were extracted with n-hexane, benzene, chloroform, ethylacetate and acetone.
The extracts were screened for their antimicrobial activity using in vitro disc diffusion method at
concentrations of 5, 2.5 and 1.25 mg/disc. The Minimum Inhibitory
Concentration (MIC) was tested using broth micro dilution method at
concentrations ranging from 5 to 0.039 mg/ml. Significant antibacterial and
antifungal activity was observed in hexane extract of flower and aerial
parts. The flower extract showed MIC as 0.15 mg/ml against Staphylococcus
aureus and the highest MIC (5 mg/ml) was noted for S. epidermidis.
The n-hexane extracts of flower and aerial parts showed MIC as 0.15 and 1.25
mg/ml respectively against Candida albicans. In conclusion, the S.
indicus flower n-hexane extract seems to be a promising antimicrobial
agent.

Different societies across the world
have shown great interest in curing illnesses using plants/plant based drugs.
Sphaeranthus indicus L. (Asteraceae)
a medicinal plant is wide spread in India and Malaysia. S. indicus has long been used in the treatment of skin
infection, bronchitis, jaundice and nervous depression (Nadkarni, 1976). The
roots and seeds are considered anthelmintic. The herb is also reported to be
useful as a tonic to treat indigestion, asthma, leucoderma and dysentery
(Chopra et al. 1956).A novel isoflavone glycoside from
leaves (Yadava and Kumar, 1999) and a new sesquiterpene glycoside and
sphaeranthanolide were isolated from the flowers of S. indicus and it was found to be an immune stimulant (Shekhani et
al. 1990).Medicinal
information from tribal healers indicated that S. indicus is used to treat skin disease, cough and fever. The
bark, ground and mixed with whey, is said to be useful in treating piles.
Flowers are credited with alterative, depurative and tonic properties; leaf
juice is boiled with milk and sugar-candy and prescribed for cough. An
aqueous extract of the whole plant was slightly toxic to American cockroaches
(Chopra et al. 1958).The present study was undertaken to
assess the antimicrobial property of the solvent extracts of flowers and
aerial parts of S. indicus.

Materials
andMethods

Plant
material

S. indicus was collected from paddy fields from Kancheepuram
district of Tamil Nadu, India. The plant was identified and confirmed by a
taxonomist and the voucher specimen (ERIB-D-73) was deposited in the
herbarium at Entomology Research Institute, Loyola College, Chennai, India.

Preparation of plant extracts

The flowers and the aerial parts were
separated, shade dried and coarsely powdered with electric blender. 200 g
powder of flowers and aerial parts were soaked separately in 600 ml of
n-hexane, in an aspirator bottle for 72 h. The extracts were collected and
concentrated at 40ºC under reduced pressure using rotary evaporator. The
extract was stored at 4ºC until further use. The remaining plant residue was
subsequently extracted with benzene, chloroform and ethylacetate similar
manner.

Test concentrations

The crude extracts were dissolved in
Dimethyl sulphoxide (DMSO) and extracts were loaded on the 6 mm dia. sterile
disc (Himedia, Bombay) with the concentrations of 1.25, 2.5, and 5 mg/disc.

The bacterial cultures were inoculated
in Mueller Hinton (MH) broth and incubated at 37ºC for 18 h at 150 rpm. The
bacterial inoculum was standardized to 0.8 OD at 660 nm and it was used for
disc diffusion method. The final inoculum size of 1x105 CFU/ml for
bacteria and 1x104 CFU/ml for Candida were used for broth micro dilution
technique. Antifungal
screening was carried out by broth micro dilution method; the final inoculum
size was 1 x 104 spores/ml.

Disc diffusion method

Preliminary antibacterial screening was
carried out using disc diffusion method (Bauer et al. 1966). Discs
with different concentrations of plant extracts were placed on the
preinoculated Mueller Hinton Agar (MHA)plates with respective cultures and were incubated at 37oC
for 24 h. Streptomycin (10 µg/disc) and DMSO were used as positive and
negative control, respectively. The inhibition zone around the disc
(diameter) was measured and recorded.

Minimum Inhibitory Concentrations (MIC)

Broth micro dilution method (NCCLS
1999) was used to determine the MIC. This was carried out in 96 well microtitre
plates containing 200 µl Mueller Hinton Broth with different concentrations
of plant extracts. The final concentration of DMSO was maintained at 0.1% in
the test broth. Triplicates were maintained along with the negative control.
Plates were incubated at 37°C for 12 h for bacteria and at 27°C for
fungi.MIC was determined as the
complete inhibition of growth at lowest concentration.

Results

Flower extracts with hexane gave pale
yellow colour and other extracts were pale brown to brown in colour. Extracts
from aerial parts were dark green to dark brown in colour. The yield of the
flower extract fell in the range of 0.5-1% (w/w) and that of aerial parts
were in the range of 1-2% (w/w) of the dried material.

Hexane extract of S. indicus (flowers and aerial parts) showed antimicrobial
activity against most of the bacteria and fungi tested. Flower extract showed
higher activity than the aerial parts against Gram positive bacteria such as B.subtilis, S. aureus, S. epidermidis and E.
faecalis which were comparable with antibiotic Streptomycin (10 µg/disc)(Table 1). The fungi Aspergillus
niger, Botrytis cinerea and C.
albicans were inhibited by the extracts of both the flower and aerial
parts. Benzene, chloroform, ethyl acetate and acetone extracts of flower and
aerial parts showed some activity at higher concentration (5 mg/disc) against
gram positive bacteria.

The hexane extract of flowers showed
MIC at 0.31 mg/ml for B. subtilis, 0.15 mg/ml for S. aureus and 5 mg/ml for S.
epidermidis. On the other hand, aerial parts showed higher MIC at 2.5
mg/ml for B. subtilis 5mg/ml for Staphylococcus
spp. and 5 mg/ml for E. faecalis
compared to flower. Most of the gram negative bacteria showed higher MIC
(>5 mg/ml) for both the extracts of flower and aerial parts (Table 3).

The flower extract showed MIC at 0.15
mg/ml and aerial parts showed MIC at 1.25 mg/ml against C. albicans; this is the first report on anti fungal activity
against Candida as per the available literature. The hexane extract of
flower showed complete inhibition against A. niger
and B. cinerae; the MIC was determined as 1.25
and 0.625 mg/ml respectively. The hexane extract of aerial parts showed MIC
as 2.5 and 0.625 mg/ml against A. niger
and B. cinerea respectively. The antifungal activity against Trichophyton spp., Epidermophyton floccosum and Microsporum cooki was reported in
thiophene compound isolated from Tagetes
patula (Asteraceae) (Romagnoli et al., 1998). Antifungal activity
was also reported for the ethanol extract of underground parts of Leuzea carthamoides (Asteraceae)
against C. albicans, A. fumigatus(Chobot et al.,
2003),and Centaurea hermanni (Asteraceae)(Sur-Altiner
et al., 1997). which is similar to
our results.

Conclusion

The S.
indicus hexane extracts of flower and aerial parts showed good
antibacterial activity against gram positive organisms. Flower extracts were
more active than the aerial parts. It also possessed strong antifungal
activity against Candida and other
tested fungi. The findings of the present research may lead to the
development of natural antimicrobial agents